Nanoparticles have unique properties that have been exploited to deliver DNA to specific animal cells. It has been found that when certain DNA-coated nanoparticles are incubated with cells not having a cell wall, the cells take up the nanoparticles and begin expressing genes encoded on the DNA. Semiconductor nanoparticles (e.g., quantum dots (“QDs”)) within the size range of 3 nm to 5 nm have also been used as carriers to deliver molecules into cells. DNA and proteins can be linked to certain ligands attached to the QD surface. See, e.g., Patolsky et al. (2003) J. Am. Chem. Soc. 125:13918. Carboxylic acid- or amine-coated QDs can be cross-linked to molecules containing a thiol group, see, e.g., Dubertret et al. (2002) Science 298:1759; Akerman et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:12617; Mitchell et al. (1999) J. Am. Chem. Soc. 121:8122, or an N-hydroxysuccinimide (“NHS”) ester group, by using standard bioconjugation protocols. See, e.g., Pinaud et al. (2004) J. Am. Chem. Soc. 126:6115; Bruchez et al. (1998) Science 281:2013. An alternative way to attach molecules to the surface of QDs is via conjugation of streptavidin-coated QDs to biotinylated proteins, oligonucleotides, or antibodies. See, e.g., Dahan et al. (2003) Science 302:442; Pinaud et al. (2004) J. Am. Chem. Soc. 126:6115; Wu et al. (2003) Nature Biotechnol. 21:41; Jaiswal et al. (2003) Nature Biotechnol. 21:47; and Mansson et al. (2004) Biochem. Biophys. Res. Commun. 314:529.
Delivery of foreign nucleic acid molecules to plants is challenging due to the presence of plant cell walls. Current methods rely on invasive delivery for genetic transformation of plants. In plant cells, the cell wall is a barrier against the delivery of exogenously applied molecules. Many invasive cell delivery methods, for example, biolisitic delivery (gene gun), microinjection, electroporation, and Agrobacterium-mediated transformation, have been employed to achieve gene and small molecule delivery into walled plant cells, but delivery of proteins has only been achieved by microinjection. Where nanoparticle delivery of nucleic acid molecules to plant cells is desired, the cell wall is stripped before the addition of the particles to protoplasts of plant. See, e.g., Torney et al. (2007) Nature Nanotechnol. 2:295-300.
Moreover, conventional plant transformation techniques, such as Agrobacterium-mediated transformation, require the use of a recombinant plasmid. These conventional techniques, therefore, result undesirably in the integration of the bacterial vector backbone sequence into the host genome along with the attached exogenous genes. See, e.g., Kohli et al. (1999) Plant J. 17:591-601; and Meza et al. (2002) Nucleic Acids Res. 30(20):4556-66. The presence of the vector backbone sequence in the transplant serves no purpose in biolistic transfer procedures. Furthermore, the vector backbone sequences have a tendency to stimulate illegitimate recombination by providing AT-rich sequences as recombination hotspots during the formation of secondary structures. Muller et al. (1999) J. Mol. Biol. 291:29-46. Vector backbone sequences may additionally produce new lengths of “filler” DNA homologous to flanking plant genomic DNA, which may escape into the environment. Kohli et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95:7203-8; Pawlowski and Somers (2000) Proc. Natl. Acad. Sci. U.S.A. 95:12106-10; Svitashev et al. (2002) Plant J. 32:433-45.
Transformation with transgene cassettes using particle bombardment has had only limited success, in tissue culture, and in rice (Oryza sativa) and potato (Solanum tuberosum). Fu et al. (2000) Transgenic Res. 9:11-9; Loc et al. (2002) Mol. Breeding. 9:231-44; Romano et al. (2003) Transgenic Res. 12:461-73; and Agrawal et al. (2005) Mol. Breeding. 16:247-60. These biolistic techniques have been suggested to generate a larger proportion of transgenic rice and potato with simple integration patterns. Two groups of linear gene constructs (GUS and bar, and 1Ax1 and bar) lacking vector backbone sequences have been independently transferred into the elite wheat (Triticum aestivum L.) variety EM12 by particle bombardment, and genetically stable transgenic plants with low copy number transgene integration were recovered. Yao et al. (2006) J. Exp. Botany 57(14):3737-46. Transformation frequency by biolistic bombardment was observed to be between 0.2 and 0.6. Id. It has been suggested that three possible elements (i.e., reducing the amount of concatemerization prior to transgene integration; limiting the occurrence of transgene rearrangements; and preventing homologous interactions between different transgenes during integration events) work together to generate simple intact transgenic loci represented by simple hybridization patterns. Agrawal et al. (2005), supra.
Particle bombardment and whiskers (See U.S. Pat. Nos. 5,464,765 and 5,302,523), together with restriction enzyme-digested DNA fragments, is the only route of delivering linear DNA cassettes to plant cells having intact cell walls at this time.